Sunflower is one of the four major oilseed crops in the world (see Carter (ed.) (1978) Sunflower Science and Technology, Agronomy Monograph 19, American society of Agronomy, Madison, Wisc.). Sunflower oil is used mainly as an edible oil and in food manufacturing. Sunflower seeds are also grown as a confectionery as well as for bird and animal feed. Significant effort has been expended in conventional breeding programs to obtain sunflower cultivars having improved agronomic characteristics, particularly those having improved oil content or quality. (see Fick (1978) in Sunflower Science and Technology, Agronomy Monograph 19, American Society of Agronomy, Madison, Wisc., pp. 279-370.) Efficient methods of sunflower regeneration are useful for facilitating breeding programs for improved cultivars by providing rapid multiplication of plants having desirable traits. The availability of a large number of regenerated plants increases the speed of selection and further breeding cycles. It is also of considerable interest to apply the methods of plant genetic engineering to develop improved sunflower cultivars. The application of these methods to sunflower improvements requires the development of methods for the efficient production of plants which have been transformed to contain foreign DNA. A number of methods are now available which allow the introduction of DNA into and selection of transformed plant cells or tissue. The regeneration of whole transformed plants from cells or tissue is often difficult or inefficient. For this reason regeneration methods that are suitable for use in combination with methods of transforming plant tissue are useful in the production of whole transformed plants.
The genus Helianthus comprises about 67 species, including the common sunflower, Helianthus annuus L. Helianthus annuus includes a number of cultivars or varieties including wild, weed and cultivated varieties (see Heiser (1978) in Sunflower Science and Technology, Agronomy Monograph 19, American Society of Agronomy, Madison, Wisc.).
The term regeneration, as used herein, relates to the vegetative or asexual formation of progeny plants from somatic cells of a parent plant. Regeneration can proceed by the induction of plant organs, particularly shoots (organogenesis) or by the induction of somatic embryos (embryogenesis) from tissues of the parent plant. Previously, shoots or embryos have typically been generated from dedifferentiated callus induced from explant tissue of the parent plant. Regeneration also refers to asexual propagation of shoots from already differentiated axillary buds in meristem, shoot tip or bud explants. This process is also called shoot multiplication or shoot proliferation. Organogenic shoots or proliferated shoots are rooted and grown into whole regenerated plants. Somatic embryos are cultured and either develop shoots which must be rooted or grow into whole plantlets with shoots and roots. Embryogenesis is distinct from embryo culture which involves the in vitro culture of zygotic embryos. Embryo culture has been used, in particular, to develop embryos resulting from interspecific crosses which often do not survive in vivo. Embryo culture provides a single plant from the cultured embryo, while embryogenesis can provide multiple embryos and plantlets from a single parent.
Several methods of sunflower regeneration have been reported. These methods include organogenesis and embryogenesis as well as shoot proliferation from meristematic tissue. A variety of explant sources have been used and a number of media variations have been employed.
Sadhu (1974) Indian J. Exp. Biol 12: 110-111 reported the differentiation of plantlets with roots and shoots from callus initiated from sunflower stem pith explants. A modified White's culture medium (White (1963) in The Cultivation of Animal and Plant Cells, Ronald Press Co, New York) (see Table 1 and Table 5) containing 1 ppm of the auxin, indole-3-acetic acid (IAA), was used. It was reported that neither 2,4-dichlorophenoxyacetic acid (2,4-D) nor kinetin supported regeneration in this system. The sunflower varieties used for this work were not described and no indication of the efficiency of the regeneration process was provided. Later reports (Paterson and Everett (1985) Plant Science 42:125-132; Cooley and Wilcox, EPO patent applications 017904 and 0171593 (filed July 9, 1985) indicate that this method is not applicable to many sunflower varieties.
Rogers et al. (1974) In Vitro 9:463 reported the establishment of callus cultures from sunflower stem explants. Explants from a single sunflower line were examined. Sunflower callus was subcultured for 7 months, then transferred to a differentiation medium containing the hormone combination: 2 mg/l IAA and 0.5 mg/l kinetin. It was reported that sunflower calli developed "tufts of hairs" at about 1 month and roots at about 3 months. Whole plants were, however, not formed from these structures.
Georgieva-Todorova et al. (1980) Proceedings of the Ninth International Sunflower Conference, Torremolinas, Spain, Vol. 1:122-128 and Bonorova et al. (1985) Z. Pflanzenzuchtg. 95: 35-44 described organogenesis of sunflower shoots from callus induced from a variety of explants, particularly pith parenchyma and stem apices. It was reported that "meristem-like" structures as well as shoots and roots were produced from cultured callus tissue, and that optimum organogenesis was obtained on MS medium (Murashige and Skoog (1962) Physiol. Plant. 15:473-497) containing 0.1 mg/l 1-naphthaleneacetic acid (NAA), 0.1 mg/l benzyl adenine (BA), 0.01 mg/l gibberellic acid (GA) and 40 mg/l adenine sulfate. However, only 1-3 shoots/explant were obtained on this medium; increases up to 3-8 shoots/explant were obtained by further addition of 800 mg/l each of L-glutamine and L-asparagine. The technique was applied to several Helianthus species and interspecific hybrids and the type of development observed was reported to be dependent on genotype as well as culture conditions.
Bohorova et al., 1985 also described attempts to regenerate sunflower via androgenesis, that is, via callus induction from anthers. It was reported that callus could be induced from anthers, however, shoot induction from such callus was unsuccessful. Direct shoot formation from anthers was reported in two cases (H. divaricatus and the interspecific hybrid from H. annuus x H. decapetalus) on a medium containing 5 mg/l zeatin. Shoots generated in this way could be used to induce callus from which more shoots could be generated.
Binding et al. (1981) Z. Pfanzenphysiol. 101: 119-130 described the regeneration of a variety of dicotyledonous plants from isolated protoplasts. Sunflower protoplasts were regenerated on V-KM agar which contained the hormones: 2.5 .mu.M BA, 5 .mu.M NAA and 0.5 .mu.M 2,4-D or on B5 medium (Gamborg et al. (1968) Exp. Cell Res. 50:151-158) containing 15 .mu.M BA. Whole sunflower plants were produced by rooting of regenerated shoot cuttings. The genotype of sunflower used and the efficiency of the regeneration were not reported.
Greco et al., (1984) Plant Science Lett. 36:73-77 reported the regeneration of sunflower plants from a variety of explants derived from seedlings. Seedlings having the first pair of non-cotyledonary leaflets were employed as the source of explants. Explants included hypocotyl segments, cotyledon parts, leaflet pieces and shoot apices. Explants were cultured on MS medium supplemented with BA and/or 2,4-D at a variety of concentrations. It was reported that cotyledon parts cultured in the presence of 2,4-D alone or in combination with BA in some cases developed callus (after about 1 month) that contained many "translucent nodules, " while cotyledon parts cultured on BA alone displayed different kinds of development which was dependent on BA concentration. Cotyledon parts cultured on 1.0 mg/l BA produced no callus, but after about 1 month, 21% of the explants produced shoots directly. It was reported that one such explant produced 26 shoots. Culture of cotyledon parts on 5.0 mg/l BA resulted in the production of callus which on reculture produced shoots. In contrast, culture of cotyledon parts on an intermediate level of BA (3.0 mg/l) induced numerous "green bulges" on the tissue surface and eventually abundant callus, but no shoots. No indication of shoot morphology was given and regeneration of whole plants from the explant induced shoots was not reported. Only one variety of sunflower (`Sannace`) was employed in these experiments.
Paterson and Everett, 1985, and Australian Patent 39152/85, filed Feb. 26, 1985, reported a method for regeneration of sunflower plants from callus induced from seedling hypocotyl explants. The medium used for callus induction and regeneration was optimized using an inbred line of sunflower and contained: MS medium (salts and vitamins) with additions of 5 g/l KNO.sub.3, 100 mg/l myo-inositol, 40 mg/l adenine sulfate and 500 mg/l casamino acids with hormones: 1 mg/l NAA, 1 mg/l BA and 0.1 mg/l gibberellic acid and 30 g/l sucrose as the carbohydrate source. The best regeneration frequency was about 9.8 shoots/hypocotyl segment. Both BA and NAA were required for successful regeneration and addition of 5 g/l KNO.sub.3 (to make a total KNO.sub.3 concentration of 6.9 g/l) greatly improved regeneration frequency. The best explants were taken from seedlings older than 5 days. Regenerability of the plants was reported to be genotype specific.
Cooley and Wilcox, EPO application 0170904, filed July 9, 1985, reported sunflower regeneration through organogenesis. The method involved culturing explants on a callus induction medium containing abacisic acid and BA, followed by subculturing callus on a shoot induction medium containing IAA and kinetin followed by subculturing shoots on a rotting medium. A related method of sunflower regeneration through embryogenesis was reported by Cooley and Wilcox EPO Application 0172377, filed July 9, 1985. This method involved culturing explants on a medium containing 2,4-D alone or in combination with abscisic acid in order to induce embryogenic callus, followed by subculturing the embryogenic callus on an embryo regeneration medium containing IAA alone or in combination with kinetin, followed by subculturing somatic embryos on a plantlet development medium. In another related method, Cooley and Wilcox EPO application 0171593, filed July 9, 1985, reported sunflower regeneration through embryogenesis and organogenesis. The multi-step method involved culturing explant tissue on a callus induction medium containing abscisic acid and 2,4-D, followed by subculturing the callus on a preconditioning medium containing BA or abscisic acid with BA, followed by subculturing the callus on a shoot formation medium containing IAA, followed by subculturing the shoots on a root induction medium. An optional callus maintenance step was also described after the initial callus induction step. In all three of these regeneration methods the preferred explant tissue was described as immature whole embryos collected at 3-7 days after pollination (embryos being less than 0.1 mm in diameter).
Trifi et al. (1981) Physiol. Veg. 19:99-102 and Paterson (1984) Amer. J. Bot. 71:925-931 reported shoot multiplication of sunflower from shoot apices and nodes of seedlings. The medium used by Trifi et al. was MS medium with additions of 3% sucrose, 0.5 .mu.g/l NAA and 0.5 .mu.g/l BA. The medium used by Paterson contained either BA or kinetin, specifically BA at concentrations between 0.1 and 1.0 mg/l and kinetin at a concentration of 1 mg/l. The efficiency of shoot multiplication also demonstrated genotype variation.
Chandler and Beard (1980) The Sunflower 6:45-47 and Crop Science (1983) 23:1004-1007 described an embryo culture system which was employed to produce interspecific sunflower hybrids. Immature embryos were excised (3 to 7 days after pollination) and cultured using a two step method: the first step, embryo enlargement, employing a medium containing low auxin levels and high sucrose concentration followed by a second embryo germination step employing a mineral salts basal medium containing low sucrose.
Paterson (1984) Amer. J. Bot. 71:925-931 reported shoot multiplication from whole or half shoot apices of H. annuus seedlings. The optimal culture medium employed as MS medium with 0.1-1.0 mg/l BA or kinetin. All inbred lines (100) tested were reported to show shoot multiplication at least one media containing 1 mg/l kinetin. A large portion (51 to 100) of inbreds tested were also reported to produce adventitious shoots on the leaves of the multiple shoots that had been induced from culture apices. In contrast, adventitious shoots could only rarely be induced by culture of seedling leaves.
The present invention provides a novel, efficient method of regenerating sunflower plants from the cotyledons of non-germinated embryos. Both mature and immature embryos can be used as explant sources, as long as the cotyledons are fully formed. The present method requires fewer steps and is more rapid than prior art methods since a separate callus induction step is not required. The present method employs a convenient explant source, sunflower seed. For many sunflower varieties mature seed can be used as the explant source, obviating the need to germinate and grow seedlings under sterile conditions as explant sources. The present method insures that substantially all of the induced shoots derive from single cotyledon cells rather than from proliferation of already differentiated multi-cellular apical buds. The present method of regeneration is generally useful for rapid multiplication and micropropagation of sunflowers and is particularly useful in combination with methods of transforming cells or tissue with foreign DNA and selecting transformed plants to obtain whole sunflower plants containing that foreign DNA.